1. Single Euploid Embryo Transfer: Fresh vs Frozen Debate
SmartArt X Slides - Charo
4/19/2017
Amber R. Cooper, MD, MSCI
Assistant Professor
Division of Reproductive Endocrinology & Infertility Washington University in St. Louis
Kaylen Silverberg, M.D.
Medical and Laboratory Director, Texas Fertility Center
Austin and San Antonio, TX
Clinical Associate Professor Dell Medical School
20-30 minute talk + 15 minutes for questions 1

2. Disclosures AC: Research Support: Beckman Coulter, Inc
Advisory Board: Celmatix, Inc.
KS:
Research grants: Actavis; Finox
Consulting/Speakers Bureaus: Good Start Genetics, Illumina, Serono, Myriad Genetics, Actavis, Abbvie

3. SHE

4. What we DO agree on….
Frozen embryo transfers are an important part of successful IVF clinics
Vitrification has improved ART success for both embryos and oocytes
Endometrial-Embryo synchrony is very important for successful implantation
PGS allows for eSET, reducing the risks associated with multiple gestation

5. FRESH OR FROZEN EMBRYO TRANSFER???
Where we differ…
What is the best FIRST line approach if you have a SINGLE EUPLOID embryo…
(assuming time/shipping barrier removed)
FRESH OR FROZEN EMBRYO TRANSFER???

6. Dr. SILVERBERG: “The Trouble with Fresh…”

7. Problems with Fresh eSET…
Effects of ovarian stimulation
Endometrial changes (advanced histology)
Premature luteinization
Dyssynchrony between embryo and endometrium
Prevents use of euploid blasts that are not vitrifiable until Day 6 or 7
Only available to programs with onsite PGS

8. Ovarian Stimulation
Controlled ovarian stimulation (COS) with exogenous FSH promotes development of multiple ovarian follicles
Multiple follicles produce supraphysiologic levels of estradiol, progesterone, and other hormones
These hormones affect and control endometrial development, maturation, and uterine contractile activity, and can alter the window of implantation
COS can lead to premature luteinization, lessening the likelihood of implantation

9. Endometrial Changes
Pinopodes appear 1-2 days earlier in cycles with COS and are less numerous
Pinopode function not yet confirmed, but theoretically have role in implantation and endometrial receptivity
Progesterone receptor down-regulated 1-2 days earlier in cycles with COS.
All of these factors are consistent with advanced endometrial histology.
…when compared to natural cycles.
Mirkin et al, Nikas et al, Develioglu et al, Horcajadas et al 2007.

10. Advanced endometrial histology
Advanced endometrial histology has been correlated with implantation failure in fresh cycles.
Nikas et al, Kolibianakis et al, 2002.

11. Overall effect of ovarian stimulation on the endometrium
Following COS, the endometrium is “histologically advanced, biochemically different, and genomically dysregulated.”
Horcajadas et al, 2007.

12. Premature Luteinization Adversely Affects Pregnancy Rates in IVF cycles

13. And it Does so by Adversely Affecting the Endometrium, not the Embryo
Retrospective evaluation (n=96)
Embryos frozen due to premature luteinization and subsequently transferred had higher pregnancy rate than did embryos frozen from cycles without premature luteinization
Silverberg K, et al. Fertil Steril 1994;61:508

14. And Freezing of Embryos with Subsequent FET can Salvage the Cycle
Retrospective cohort study, n=236 cycles
All with Premature luteinization
118 with fresh ET, 118 with freeze all and FET
Freeze-all resulted in better outcomes:
Implantation rates (56.8 vs. 26.9%, P<0.05)
Ongoing pregnancy rates/transfer (70.5 vs. 35.2%, P<0.05)
Shapiro B, et al. Fertil Steril 2010;93:636-41

15. Cryopreservation rescues cycles with “premature luteinization”
The cancellation rates did not differ significantly (P=0.093). All other rates shown differed significantly. The difference in pregnancy losses was mainly in biochemical pregnancy losses, which differed significantly (4/81 in FET vs 15/62 in fresh transfer, per pregnancy, P=0.0010).
Shapiro et al 2010, comparing 236 matched cycles with elevated P4.

16. Embryo developmental pace
There is biological variation in embryonic development
Some embryos form expanded blastocysts on day 5 of development, others on day 6 or 7
Day 5 and 6 blastocysts are more likely to be chromosomally normal than day 7 blastocysts in fresh IVF cycles following ovarian stimulation.
Silverberg K et al, 2015.

17. Embryonic Euploidy
Nguyen J, Silverberg K, et al, 2014.

18. Embryo developmental pace
Shapiro et al 2001.

19. Therefore:
Some (Day 6, Day 7) euploid blastocysts won’t even be available for fresh ET, as fresh ET must occur on Day 5
In addition:
Vitrification allows for embryo batching/banking, and is available to labs that do not have onsite PGS capability
MOST IMPORTANTLY:
Vitrification with subsequent FET allows for TRULY optimal embryo selection that you CAN’T achieve with fresh ET

20. Vitrification with FET Affords Pregnancy Rates At Least As Good As Fresh ET

21. Pregnancy Rates in Fresh, Frozen, and donor oocyte cycles
Retrospective study:
377 fresh autologous cycles
106 autologous FET cycles
56 fresh oocyte donation cycles
Shapiro et al 2008.

22. Contrasting patterns of clinical pregnancy rates in fresh and FET
Shapiro et al 2008.

23. Can FET in young patients be comparable to fresh donor cycles?
Advantages of donor oocyte cycles:
transfer of healthy embryos derived from young oocytes
Absence of an endometrium exposed to supraphysiolgic hormone levels resulting from COS
Therefore, shouldn’t the implantation and pregnancy rates of young patients in FET cycles rival those of donor oocyte cycles?
69 non-donor FET using PTEC
136 fresh oocyte donation cycles
Shapiro et al 2010.

24. Autologous FET (136) vs. fresh (69) oocyte donation cycles
How does FET in young patients compare to fresh donor cycles using young donors?
Autologous FET (136) vs. fresh (69) oocyte donation cycles
All patients and donors <35 yo
Implantation rates (65.9% vs 62.1%)
Ongoing pregnancy rates (79.7% vs 75.0%).
69 non-donor FET using PTEC
136 fresh oocyte donation cycles
Shapiro et al 2010.

25. The passenger was on time, but the train left early

26. Randomized Trial: Fresh vs Frozen in Normal Responders
Randomized trial comparing fresh and frozen embryo transfers in 103 NORMAL responders (8-15 antral follicles) age years.
Shapiro et al 2011.

27. Randomized Trial: Fresh vs Frozen in Normal Responders
Cryo
P-value
Implantation rate
37/95 = 38.9%
63/89 = 70.8%
<0.0001
Clinical pregnancy rate per transfer *
29/53 = 54.7%
42/50 = 84.0%
0.0013
Ongoing pregnancy rate per transfer
27/53 = 50.9%
39/50 = 78.0%
0.0072
* The study was halted at this interim stopping point because the P-value was less than 0.03, per the pre-defined stopping rule.

28. Fresh versus Frozen IVF Outcomes
When compared to fresh transfer, embryo cryopreservation followed by FET has been associated with:
Reduced risk of implantation failure in normal responders
Reduced risk of implantation failure following premature luteinization
Reduced risk of IVF failure per retrieval
Shapiro et al 2011, Shapiro et al 2010, Roque et al 2012

29. Preliminary Conclusions -Why Vit/Thaw is UNQUESTIONABLY SUPERIOR…
Avoid potential adverse effects of COS on endometrium
Avoid effects of premature luteinization
No need for in house PGS lab
More euploid embryos available for use
Better perinatal/neonatal outcome (as you will see)

30. Dr. Cooper: “The Trouble with Frozen…”

31. So let me get this straight…

32. WHAT DID YOU JUST HEAR? Endometrial-Embryo synchrony is very important
Pay attention to method and degree of COH
Early progesterone elevations may be detrimental
Good quality day 5 blasts have much better implantation rates than day 6 in fresh cycles; Those that are slower growing may do better in a frozen cycle

33. So….SLOW DOWN THE TRAIN !! (or at least put it on the right schedule)

34. Maybe we should focus on WHY there is dyssynchrony
Too much drug?
Hyperstimulation?
Type of stimulation?
Advanced follicular development, delayed trigger?
Do we need a more physiologic trigger?
Improved monitoring of the luteal phase?

35. Do we agree on which FET approach is best?
Timing of embryo cryopreservation?
2PN, CD2/3, CD5/6
Slow-freezing vs vitrification?
Medicated or natural cycle ET? Leuprolide?
Recommended number of embryos to transfer?
Culture media?
Timing and delivery method of progesterone?
So, how do we really look at outcomes critically? And as Dr. Scott says…

36. Slow-freezing vs Vitrification: Vitrification data still limited
No perinatal risks D3 embryos
Liu SY, Hum Reprod 2013
Shi W, Fertil Steril 2012
No perinatal risks D5/6 embryos
Takahashi K, Fertil Steril 2005 (no diff congenital defects)
Wikland M, Hum Reprod 2010 (vit blastocyst vs D3 slow)
The majority of perinatal risk data is from slow freezing and cleavage stage embryos
Yet the majority of practice is vitrified blasts
About 4000 babies first couple studies

37. Is extended embryo culture ideal?
Yes, for improving implantation and pregnancy rates, and minimizing higher order multiples
BUT D5/6 transfer as compared to D3 increases the risk of Preterm Delivery by 30-40%
US: Kalra S et al. Obstet Gynecol 2012; SART data, 69,039 LB, fresh ET
Canada: Dar S et al. Hum Reprod 2013; 12,712 LB
Sweden: Kallen B et al. Fertil Steril 2010; 13,873 LB
D5/6 vs D3 also with increased absolute birthweight and incidence of LGA
China: Zhu J et al. Hum Reprod 2014; 2,929 singletons, fresh,
Makinen S, Hum Reprod 2013
2/3 CD3; SART
Fresh, no diff in LBW %
China study-difference pt bias, initial tx d3 tfr; day 5/6 if past implantation failure

38. An additive (or masking) effect?
Ovarian Stimulation
…In subtle epigenetic effects…?
In vitro culture
Extended Culture
…Is cryo just one more piece…?
Cryo-
Preservation

39. The forest through the trees…
Subtle differences at birth may
have health consequences that
manifest as adults
Barker hypothesis
Grace and Sinclair 2009 (animal review)
Ceelen M, 2008; higher BP/FBG
8-18 y/o ART kids
Hart and Norman 2013; higher BP/FBG in ART adults
We need to sort out the underlying etiologies

40. Other issues with FET Additional cost Additional time
Disposition of excess embryos
Optimal length of storage for vitrification?
The lab is critical…
What about transferring embryos between centers?

41. And do all patients really need and/or benefit from aneuploid screening?
I concede….some may
May improve eSET in some patients
But…well selected patients may benefit from eSET without AS (at least in our clinic)
Much of success relies on TE biopsy and advanced genetic testing
Are all abnormals abnormal? 10-12% may not be
What about the 10+% that don’t have blastocysts develop?

42. Risks to Mom and Baby…

43. Fresh versus Frozen IVF Outcomes
When compared to fresh transfer, embryo cohort cryopreservation followed by frozen-thawed transfer has been associated with:
Reduced risk of implantation failure in normal responders
Reduced risk of implantation failure following premature progesterone elevation
Reduced risk of IVF failure per retrieval
Shapiro et al 2011, Shapiro et al 2010, Roque et al 2012

44. Fresh versus Frozen Maternal Risks
When compared to fresh transfer, frozen-thawed transfer has been associated with:
Reduced risk of late-onset OHSS
Reduced risk of ectopic pregnancy
Reduced risk of pre-eclampsia.
ASRM Practice Committee 2008, Ng et al, Ishihara et al, Shapiro et al, Maheshwari et al 2012, Imudia 2013.

45. Perinatal Risks related to Birthweight
Fresh versus Frozen
Perinatal Risks related to Birthweight
When compared to fresh transfer, frozen-thawed transfer has been associated with:
Greater mean birthweight
Reduced risk of low birthweight
Reduced risk of small for gestational age.
Compared with fresh transfer:
Wang (2005) reported reduced risk of low birthweight and prematurity.
Shih (2008) reported greater birthweight with frozen embryos.
Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption.
Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight.
Henningsen (2011) reported greater birthweight with fresh than with FET.
Kalra (2011) reported reduced perinatal morbidity.
Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight.
Imudia (2012) reported reduced risk of SGA and pre-eclampsia.
Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption.
Nakashima (2012) reported greater birthweight and reduced risk of low birthweight
Sullivan (2013) reported reduced risks of prematurity and perinatal death.
Pinborg (2013) reported reduced risk of prematurity.
Maheshwari et al (2012)

46. Perinatal Risk of Pre-Term Delivery
Fresh versus Frozen
Perinatal Risk of Pre-Term Delivery
When compared to fresh transfer, frozen-thawed transfer has been associated with:
Reduced risk of pre-term birth
Reduced risk of pre-term low birthweight
Compared with fresh transfer:
Wang (2005) reported reduced risk of low birthweight and prematurity.
Shih (2008) reported greater birthweight with frozen embryos.
Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption.
Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight.
Henningsen (2011) reported greater birthweight with fresh than with FET.
Kalra (2011) reported reduced perinatal morbidity.
Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight.
Imudia (2012) reported reduced risk of SGA and pre-eclampsia.
Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption.
Nakashima (2012) reported greater birthweight and reduced risk of low birthweight
Sullivan (2013) reported reduced risks of prematurity and perinatal death.
Pinborg (2013) reported reduced risk of prematurity. .
Maheshwari et al 2012, Kalra et al 2011, Sullivan et al 2013, Pinborg et al 2013

47. Risks Associated with Pre-Term Delivery
Inability to regulate body temperature
Respiratory distress or apnea
Visual issues, including retinopathy
Feeding problems, digestive issues
Prolonged hospitalization
Intellectual disabilities
Low birthweight
Hearing loss
Jaundice
Bleeding in the brain
Infection
Cerebral palsy
Neonatal death

48. Fresh versus Frozen Other Perinatal Risks
When compared to fresh transfer, frozen-thawed transfer has been associated with:
Reduced risk of antepartum hemorrhage
Reduced risk of placenta previa
Reduced risk of placental abruption
Reduced risk of perinatal mortality
Compared with fresh transfer:
Wang (2005) reported reduced risk of low birthweight and prematurity.
Shih (2008) reported greater birthweight with frozen embryos.
Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption.
Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight.
Henningsen (2011) reported greater birthweight with fresh than with FET.
Kalra (2011) reported reduced perinatal morbidity.
Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight.
Imudia (2012) reported reduced risk of SGA and pre-eclampsia.
Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption.
Nakashima (2012) reported greater birthweight and reduced risk of low birthweight
Sullivan (2013) reported reduced risks of prematurity and perinatal death.
Pinborg (2013) reported reduced risk of prematurity.
Maheshwari et al 2012, Sullivan et al 2013

49. Risks to Mom and Baby…

50. Fresh vs Frozen: Perinatal risks
IF you have euploid blasts to work with, IF they are recovered, IF they survive, and IF they are of optimal quality
Numerous cohort and meta analyses suggest outcomes better with FET as compared to fresh (SGA, LBW, PTD) but still worst than spontaneous
Pelkonen 2010, Pinborg 2010, Sazonova 2013, Wennerholm 2009, Maheshwari 2012, Pinborg 2012
No difference in birth defects in fresh vs FET
Meta-analysis: Maheshwari A, Fertil Steril 2012; OR 1.1 ( )
Hansen M Obstet Gynecol 2012, Davies NEJM 2012, Jalliday JL Hum Reprod 2010
SGA, LBW and PTD often better with FET
-most data with slow-freezing

51. Fresh vs Frozen: Perinatal risks
BUT, Numerous studies suggest increased risk of LGA and macrosomia with FET vs fresh or spontaneous
Pelkonen 2010, Pinborg 2010, Sazonova 2012
Wennerholm 2013: Nordic cohort, CoNARTaS group: 6647 FET, fresh, spontaneous singletons-increased risk LGA, macrosomia, and perinatal mortality (49% increase)
Wennerholm 2013, most slow freeze, day 2, txfr natural cycle
Perinatal risk: stillbirth> or equal 28 wks to neonatal death
Adj for mat age, parity, year of birth, gender, country

52. Perinatal risks: LGA
Is the epigenetic regulation of the placenta altered?
Nelissen 2011, Vergouw 2012
Denmark study FET vs fresh, sibling subgroup 2-4 fold higher incidence of LGA in FET siblings; Pinborg 2014
Is it in vitro culture media/methods?
Young 1998 (cattle/sheep), Dumoulin 2010, Nelissen 2012
In mice imprinting changes with in vitro culture more pronounced with freezing; Market-Velker 2010
Is it the extended culture issue?
Makinen 2013
An endometrial synchrony issue?
Wilmut I 1981; D3 sheep embryos transferred to advanced lining caused 40% increased fetal size
Pinborg 2014, >99% FET slow frozen CD2

53. Perinatal risks: LGA More recent vitrification data
Kato et al, 2012; Japan ( cycles)
4092 vitrified SET vs 2531 fresh SET
Transfers D2 and D5
LGA crude OR 1.51 ( ), aOR 1.23 ( ) after adjustments including blastocyst culture
D3 fresh vs D3 vitrified-no difference perinatal outcomes in general
Liu SY et al 2013, Shi W et al 2012

54. And for SGA…?
Fresh may have increased risk of SGA as compared to frozen
Helmerhorst 2004, Jackson 2004, McDonald 2009
Though underlying infertility etiology and supraphysiologic levels of hormone may be more to blame…
Cooper A 2011 Fertil Steril
1200 vs 460 F vs I; infertile had smaller neonates and more LBW than fertile at birth and in the first trimester; Within infertiliy subgroups no differences

55. Wash U fresh vs frozen sibling study
CRL did not differ between the groups (difference 1.3 mm, 95% CI -1.1 to 3.7)
No difference in birthweight between fresh & FET singleton siblings (difference 129 grams, 95% CI -155 to 412)
FET singletons had a significantly shorter gestation than their fresh IVF siblings, delivering on average 14 days earlier (95% CI 3-25 days; p=0.01)
Fresh (n=23)
Frozen (n=23)
p value
Crown Rump Length
(millimeters)
8.3 ± .8
7.3 ± 1.0
p =.86
Birthweight
(grams)
3280 ± 100
3248 ± 158
p = .87
Gestational Age at Birth
(days)
252 ± 3
241 ± 4
p = .01
Primary outcome were size (measured in the 1st trimester with CRL in mm and at birth with weight in grams) and gestational length measured in days.
O’Neill KE and Cooper AR. Fertil Steril 2010; 94(4); S19

56. Perinatal risks: Neurodevelopment?
Swedish study, births (2.5 million infants, 1.2% by IVF)
IVF vs spont, ICSI vs no ISCI, fresh vs frozen
No increased risk of autism in fresh vs frozen, ICSI vs no, only with surgically extracted sperm
aRR MR if fresh + ICSI 1.47 ( ), ICSI + frozen + preterm birth 3.47 ( )
When adjusting for singletons only ICSI + frozen remained significant for MR; 2.36 ( )
Rate per 100,000 person years. AR small
Evolving theories of epigenetics in brain development
Incidence of autism and MR 1-1.5%
Risk of MR in IVF vs spont disappeared w singletons only
Blastocyst data available for 2002+, subanalysis no difference though smaller numbers
Sandin S et al, JAMA 2013

57. SART TRENDS…

58. Trends in Fresh and FET outcomes
SART registry
Standard SART age groups
Compared with fresh transfer:
Wang (2005) reported reduced risk of low birthweight and prematurity.
Shih (2008) reported greater birthweight with frozen embryos.
Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption.
Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight.
Henningsen (2011) reported greater birthweight with fresh than with FET.
Kalra (2011) reported reduced perinatal morbidity.
Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight.
Imudia (2012) reported reduced risk of SGA and pre-eclampsia.
Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption.
Nakashima (2012) reported greater birthweight and reduced risk of low birthweight
Sullivan (2013) reported reduced risks of prematurity and perinatal death.
Pinborg (2013) reported reduced risk of prematurity.

59. Trends in US National Average Live Birth Rates

60. Trends in US National Average Live Birth Rates

61. Trends in US National Average Live Birth Rates

62. Trends in US National Average Live Birth Rates

63. Trends in US National Average Live Birth Rates

64. Trends in numbers of live births
Values calculated from SART national reports.
Fresh live births are products of live birth per cycle and number of cycles.
FET live births are product of live birth per transfer and number of transfers.

65. BUT…
…I expect the 38+ year old who HAS blastocysts to freeze to do better than her age matched friends

66. BUT…
It goes back to what is our FIRST line therapy for a single euploid blastocyst…
There is a place for FET
There is room for improvement in fresh cycles
We need more research on vitrified blasts (and oocytes)
We need more research on extended culture (would outcomes look as good with day 2/3 freeze?)
Genetic testing is good but not perfect
If time and money weren’t an issue…
And if only humans were efficient reproducers…

67. Summary of arguments
Fresh
Most cryopreservation done at blast stage, so arguments against blast transfer are potentially valid; success is based on predominantly blast transfers
Hypothetical additional epigenetic concerns
Increased cost of cryopreservation and subsequent FET

68. Summary of arguments Frozen
Cryopreservation can be done at MC stage with cleavage stage or blast transfer
Only hypothetical epigenetic concerns to date
Lower costs from:
Increased implantation and pregnancy rate
Savings in elimination of multiples
Avoiding FETs of non-viable embryos
Fewer cycles required for pregnancy

69. Summary of arguments
Fresh
Loss of embryos from freeze/thaw
PGS and the transfer of euploid embryos may negate any lowered implantation rates in fresh cycles
Larger babies therefore higher cesarean section rates and potentially long term health consequences of faster/larger growth
Convenience from completing retrieval and transfer in one cycle (TIME is important to aging women!)

70. Summary of arguments
Frozen
Avoids wasting good embryos by not transferring into a suboptimal endometrium
Higher implantation rate increases success with single embryo transfer
Lower risk of preterm birth and low birth weight babies
Convenience of scheduling subsequent FET
Opportunity to freeze and wait for PGS results
“Aging” women prefer better results

71. Thank you!